布洛芬降解菌生长特性研究及降解基因筛选

发布时间：2018-05-06 14:28

本文选题：布洛芬 + 微生物降解　；参考：《西北农林科技大学》2015年硕士论文

【摘要】：布洛芬在为人类减轻疼痛的同时也带来一定的环境污染,为了缓解此环境问题,以筛选到的3株布洛芬降解菌为研究对象,进行布洛芬的微生物降解研究,以期为布洛芬污染的微生物修复提供理论与实践依据。将从西安污水处理厂水样分离到的3株以布洛芬为唯一碳源的降解细菌进行菌株鉴定与生长特性研究,分别构建基因文库以筛选布洛芬降解基因,成功构建3株细菌的福斯质粒文库后筛选得到1株可以产生褐色邻苯二酚代谢物的阳性克隆菌株,再次构建该克隆菌株的突变体文库,筛选得到7株不再积累褐色代谢物的突变体菌株,分别对功能缺失克隆进行测序分析,初步确定布洛芬降解菌的降解相关基因。主要研究结果如下:(1)分别通过对3株布洛芬降解菌的显微形态学观察,生理生化特性实验,16S rDNA鉴定及构建系统发育树对其进行种属的鉴定,最终确定I-2为克雷伯氏菌属(Klebsiella sp),I-4为假单胞菌属(Pseudomonas sp),I-14为不动杆菌属(Acinetobacter beijerinckii)。(2)分别通过生长特性实验对3株布洛芬降解菌进行最适生长条件的探索,结果显示3株菌的最适培养温度均为30℃,I-2,I-14最适pH为7,而I-4最佳pH是6,对于布洛芬底物浓度的耐受I-2最高,为1000 mg/L,I-4次之,耐受浓度是900 mg/L,I-14最低,但也达到500 mg/L。(3)分别提取3株菌株的基因组DNA构建了3个菌株的福斯质粒文库,通过对文库的筛选在I-2福斯质粒文库中得到1株能够积累邻苯二酚代谢物的阳性克隆2H10,该阳性克隆可以利用布洛芬为唯一碳源生长,说明其含有布洛芬降解基因。(4)对含有降解基因的阳性克隆菌株2H10构建Tn5插入突变体文库,经过对该突变体文库的筛选确认得到7株功能丧失菌株,对这些菌株进行测序分析,筛选到五个编码基因,初步确定I-2的降解相关基因。
[Abstract]:Bloven also brought certain environmental pollution to alleviate the pain for human beings. In order to alleviate this environmental problem, 3 strains of Bloven degrading bacteria were selected as the research object, and the microbial degradation of Bloven was studied in order to provide the theory and practice basis for the microbial remediation of Bloven pollution. The water samples from the Xi'an sewage treatment plant will be provided. The isolation of 3 strains of biodegradable bacteria with ibuprofen as the only carbon source were identified and the growth characteristics were studied. The gene library was constructed to screen the ibuprofen degrading gene, and 3 strains of bacterial plasmid library were successfully constructed. 1 strains of positive clones that could produce brown catechol metabolites were screened, and the clone was constructed again. 7 mutant strains that no longer accumulated Brown metabolites were screened, and the functional deletion clones were sequenced and analyzed. The main results were as follows: (1) the micromorphological observation and physiological and biochemical characteristics of 3 strains of ibuprofen degrading bacteria were observed respectively. 16S rDNA identified and constructed phylogenetic tree for identification of its species, and finally identified I-2 as Klebsiella (Klebsiella SP), I-4 as Pseudomonas (Pseudomonas SP), I-14 as Acinetobacter (Acinetobacter beijerinckii). (2) exploration of the optimum growth conditions for 3 strains of ibuprofen degrading bacteria through growth characteristics experiments. The results showed that the optimum culture temperature of 3 strains were 30, I-2, I-14 was 7, and the best pH of I-4 was 6. The highest tolerance to ibuprofen substrate concentration was I-2, 1000 mg/L, I-4 time, 900 mg/L and I-14 lowest, but 500 mg/L. (3) also obtained 3 strains of genomic DNA to construct the FOSS plasmid library of 3 strains, respectively. By screening library, 1 positive clones that could accumulate catechol metabolites were obtained in I-2 FOS plasmid library. The positive clones could be grown with ibuprofen as the only carbon source, indicating that it contains the ibuprofen degradation gene. (4) the Tn5 insertion mutant library was constructed for the positive clon strain 2H10 containing the degrading gene, and the Tn5 inserted mutant library was constructed. The mutant library was screened and confirmed to obtain 7 strains of functional loss. The strains were sequenced and analyzed, and five encoding genes were screened to determine the related genes of I-2 degradation.

【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X172

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